Development and bioreactor culture of collagen-hydroxyapatite scaffolds for bone tissue engineering
Niamh A. Plunkett1,2, John P. Gleeson1,2, Michael J. Jaasma1,2, Fergal J. O’Brien1,2
1. Department of Anatomy, Royal College of Surgeons in Ireland, Dublin.
2. Trinity Centre for Bioengineering, Trinity College, Dublin.
Abstract
Tissue engineering provides an alternative solution for bone graft substitution to the standard, problematic
treatments of autografts and allografts. Using a lyophilisation process, this study has developed a scaffold
for bone tissue engineering from the two primary constituents of bone, collagen and hydroxyapatite (HA).
Four scaffold variants were produced using increasing amounts of HA. The resulting scaffolds were highly
porous (over 99%), permeable, supported cell growth and exhibited mineralisation after 28 days in culture.
The scaffold with the highest percentage of HA present was chosen as the most promising for bone tissue
engineering and was seeded with pre-osteoblastic cells and cultured in a flow perfusion bioreactor
developed in our laboratory to enhance cell distribution and mechanically stimulate the cells. Bioreactor
culture was found to enhance cell distribution in comparison to statically cultured constructs. The results
demonstrate that using the bioreactor in combination with the novel scaffold may provide a promising
strategy for bone tissue engineering.
Introduction
Limitations with autografts and allografts, the most commonly used grafts in bone replacement, include
expense, limited size of obtainable graft, donor site morbidity, potential for rejection and complications
arising from infection and chronic pain [1]. Due to these problems, interest in alternatives such as tissue
engineering solutions has increased. Bone tissue engineering involves seeding bone cells onto a scaffold,
culturing this construct so that mineralisation occurs (by using signalling mechanisms such as growth
factors or bioreactors) and then implanting it into a defect site in the body [2]. The scaffold must provide a
framework and yet have an open-pored structure that allows nutrients to penetrate into the scaffold in vitro
and then vascularisation to occur in vivo. A pore volume fraction (porosity) of over 90% is desirable in
order for cells to be viable in the construct [3; 4]. Another important parameter, permeability, depends on a
number of factors, including porosity, pore size/distribution and pore interconnectivity [3] and governs how
easy it is for nutrient delivery to and waste removal from the centre of the construct to occur. This then
governs whether the scaffold is a suitable environment for cells to live in and grow. Here, composite
scaffolds are presented that are fabricated from collagen and HA, the major constituents of bone. The aim in
combining a polymeric material (collagen) and a ceramic one (HA) in a composite material is to combine
the advantages of both materials, while overcoming any drawbacks they may have. Thus, the polymer
imparts flexibility to the scaffold while the ceramic provides a reinforcing effect. The fabrication process
(lyophilisation) used is highly effective in composite material fabrication, as well as being highly
controllable and enabling highly porous, homogeneous scaffolds to be produced [5].
A major setback to bone tissue engineering has been the inability to develop homogeneous tissue engineered
constructs in static culture. Bioreactors can be used to overcome this problem. Flow perfusion bioreactors
force fluid through constructs, thus delivering nutrients to the centre of constructs, where necrotic regions
occur, and exerting a shear stress on the cells which can mechanically stimulate them. A flow perfusion
bioreactor has been designed and validated in our laboratory [6]. Use of this bioreactor has shown that rest
periods of 7 hours between bouts of stimulation allow cells to restore their mechanosensitivity so that they
can respond again to flow [7] and that short term rest periods of the order of seconds are also useful in
restoring mechanosensitivity. The goals of this study were to develop and characterise collagen–
hydroxyapaptite (CHA) scaffolds for use in bone tissue engineering and to use the optimal scaffold in the
flow perfusion bioreactor in an effort to enhance cell distribution and mechanically stimulate the cells on the
construct.
Materials and Methods
Scaffold fabrication and analysis: Type I bovine collagen (Collagen Matrix, USA) was blended with HA
(Plasma Biotal, UK) in acetic acid to form slurries. CHA scaffolds were fabricated by freezedrying these
slurries over 24 hours using a final freezing temperature of -40°C [5; 8]. Four different scaffolds were
produced: (i) control collagen-only scaffolds (0% HA) (ii) 50wt%HA scaffolds (2:1 ratio of collagen to HA)
(iii) 100wt%HA scaffolds (1:1 ratio) and (iv) 200wt%HA scaffolds (1:2 ratio). Scaffolds were crosslinked
via dehydrothermal crosslinking in a vacuum oven at 120°C and 0.05 Bar for 24 hours. Scaffold discs (12.7
mm in diameter) were further crosslinked using EDC/NHS (14 mM N-(3-Dimethylaminopropyl)-N’ethylcarbodiimide hydrochloride and 5.5 mM N-Hydroxysuccinimide in distilled water; Sigma-Aldrich,
Germany) [9]. Porosity was examined by measuring the density of the dry scaffold samples and comparing
this to the density of the materials from which the scaffolds were fabricated. The permeability of scaffold
samples was obtained by using a permeability rig designed and fabricated in-house. 12.7 mm diameter
samples were placed into the rig under a column of water which was allowed to flow through the samples
for 5 minutes. Darcy’s law was then used to obtain the permeability as follows:
k  Qh AP where k is the hydraulic permeability in m 4 / Ns , Q is the volume flow rate in m 3 / s , h is the
height of the scaffold, A is the cross sectional area of the flow path and P is the pressure exerted by the
column of water. A sample size of at least five per scaffold type was used. The Young’s modulus in
compression of the scaffolds was obtained using a mechanical testing machine (Z050, Zwick/Roell,
Germany) fitted with a 5-N load cell. Testing was carried out in a bath of PBS at a strain rate of 10% per
minute to a maximum strain of 10%. The modulus was defined as the slope of a linear fit to the stress-strain
curve over 2-5% strain [10]. A sample size of at least 10 per scaffold type was used.
Scaffolds were seeded with 2 million MC3T3 E1 cells and cultured for 7, 14, 21 or 28 days. A sample size
of 4 per scaffold type per time point was used. Cell number was quantified using Hoechst 33258 (SigmaAldrich, Germany). Cell distribution was examined by wax embedding scaffold samples, slicing at 10µm on
a mictotome (Leica Microsystems, Germany), staining using haematoxylin and eosin and examining under a
light microscope (Nikon, Japan). Mineralisation was examined by staining using alizarin red s and
quantified
using
cetylpyridinium
chloride
(Sigma-Aldrich,
Germany)
[11].
Bioreactor culture: The 200wt%HA scaffold which proved to have the best osteogenic potential (see
Results) was seeded with 2 million MC3T3 E1 cells, and cultured for 6 days. Subsequently, scaffolds were
either cultured statically for a further 169 hours (Static group) or placed in the flow perfusion bioreactor.
The flow perfusion bioreactor designed and validated in our laboratory consists of a programmable syringe
pump (New Era Pump Systems Inc., USA), a scaffold chamber and a reservoir [6]. The constituent parts are
connected via silicone tubing. This provides a closed circuit in which media can be circulated (Figure 1). 6
scaffold chambers were used in each experiment and each experiment was run at least twice to ensure
repeatability. Of the groups placed into the bioreactor, two were controls and two experienced flow. The
two controls were (i) a Timeless group which was placed into the bioreactor and then immediately removed,
to assess the effect this process had and (ii) a Control-169 group which remained in the bioreactor for 169
hours but did not experience flow. Each flow group used 1 hour of stimulation followed by a resting period
of no flow for 7 hours, with this pattern being looped for the entire culture period of 169 hours. The two
flow groups were (i) a Steady group which experienced flow at 1mL/min for the stimulation period (ii) a
Rest-Inserted group in which short-term periods of no flow of duration 5 seconds were incorporated after
each 10 seconds of 1mL/min flow in the stimulation period, as this can restore mechanosensitivity to
desensitised cells. Cell number and cell distribution were examined as above. Statistical analysis was done
in Minitab 15 (Minitab Inc., USA) using general linear model ANOVA and the Tukey test as the post-hoc
test.
A
Media
Reservoir
Scaffold
Chamber
Syringe Pump
Figure 1. Diagram of flow perfusion bioreactor. ‘A’ shows one of the 6 systems that make up the bioreactor
[6]. ‘B’ shows a closer view of a scaffold chamber.
Results
Scaffold fabrication and analysis: Porosity values ranged from 99.5% ±0.03% for the control collagenonly scaffolds to 99.0% ±0.06% for the 200wt%HA scaffolds. Permeability values increased with
increasing HA content, to a maximum of 4.8 x10-9 m4/Ns ±1.5 x10-9 m4/Ns for 200wt%HA scaffolds. The
Young’s modulus also increased significantly with HA content, with the 200wt%HA scaffold having a
compressive modulus of 3 kPa ±1.6 kPa compared to the collagen-only scaffold’s modulus of 1.6kPa ±1
kPa (p<0.00005). Cells were viable on all scaffolds at 28 days, with all CHA groups showing an increase in
cell number over the culture period of 10-48% while the control collagen-only group experienced a slight
reduction in cell number of 13% (Figure 2). 50wt%HA scaffolds showed low initial cell attachment but
good proliferation of cells. Mineralisation occurred at the surface of all scaffolds by day 28 (p=0.0137), with
quantifiably more mineralisation occurring on the 200wt%HA scaffold group than any other group
(p<0.0021).
*
Cell number (million)
4
*
3.5
3
c
7 day
14 day
21 day
28 day
c
2.5
2
1.5
1
0.5
0
collagenonly
50wt%HA
100wt%HA
200wt%HA
Figure 2. Cell number on the four scaffold types over a 28 day culture period. Average cell number
increased on all CHA scaffolds over the culture period, while it decreased on the collagen-only group (these
changes were not statistically significant). All groups had higher cell number than the 50wt%HA group (*
represents p<0.0016), although this group experienced the greatest amount of proliferation over the culture
period.
Bioreactor culture: A trend for cell number reduction due to bioreactor culture was noted but was found to
be non-significant and a substantial number of cells were retained on the constructs after the culture period
(Figure 3, p=0.081). 1.3 – 1.4 million cells were attached to the flow group constructs after 169 hours of
bioreactor culture, a large proportion (65-70%) of the amount initially seeded. Cell distribution was
enhanced due to flow, as can be seen in the images in Figure 4. The static group exhibited a cellular
distribution that was predominantly on the periphery of the construct, while in the flow groups the cells
were dispersed in a homogeneous manner throughout the construct.
Cell number (millions)
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
timeless
static
control-169
steady
rest-inserted
Figure 3. Cell number on groups used in the bioreactor study. No significant difference was found between
groups, but there was a trend for bioreactor cultured groups to have a lower cell number compared to the
static culture groups (p=0.081).
Static
Rest-Inserted
Figure 4. Images of haematoxylin and eosin stained longitudinal sections of a static group construct (on left)
and a rest-inserted group construct (on right). Cells reside predominantly on the periphery of the static group
constructs while they are dispersed homogeneously throughout the flow group constructs. Scale bars are
200µm in length.
Discussion
This study has led to the development of patented, novel composite scaffolds with significant potential for
bone repair and, furthermore, has shown that use of a flow perfusion bioreactor improves cellular
penetration into the scaffolds. The CHA scaffold and the process for its production was submitted to the
European Patent Office (EPO) in February 2008 and published internationally under the Patent Cooperation
Treaty (PCT) in August 2008 [8]. Inclusion of HA in the scaffolds increases scaffold stiffness and
permeability while retaining the very high porosity, and significant advantageous properties, of collagenonly scaffolds. The fabrication technique (lyophilisation) utilised offers a large degree of architectural
control, providing scaffolds which should meet all of the criteria of an osteoconductive scaffold. High
porosity is important to enable cell infiltration into the scaffold, and a high permeability allows for better
nutrient exchange and waste removal from the scaffold. The permeability of trabecular bone is reported as
being from 10-6 to 10-12 m4/Ns and the scaffold permeability lies midway within this wide range, which is a
very promising result [12]. The scaffolds are highly biocompatible, showing an ability to support cell
growth to 28 days in static culture, with modest cell number increases being noted on all CHA scaffolds. In
addition, mineralisation of the scaffolds occurred by day 28, with significantly more mineral deposition on
the 200wt%HA constructs than on any other group. This ability to enable mineralisation to occur
demonstrates the osteogenic potential of the CHA scaffold, a potential which is vital for its use in bone
tissue engineering. The 200wt%HA scaffold exhibited a high porosity of 99%, the highest permeability of
the scaffold types tested and the highest compressive stiffness, thus pinpointing it as the most promising
scaffold from a structural perspective. Furthermore, the biocompatibility of the 200wt%HA scaffold was
similar to the highly biocompatible collagen scaffold and its mineralising ability surpassed all other scaffold
types tested. Due to this promising combination of structural and osteogenic properties, the 200wt%HA
scaffold was chosen for use in further experimentation in a flow perfusion bioreactor. Use of the bioreactor
in conjunction with this novel scaffold has enabled better cell distribution throughout the scaffold to be
obtained. While constructs cultured statically exhibited a cellular distribution where cells resided
predominantly on the periphery of the construct, flow group constructs that had been cultured in the
bioreactor for 169 hours displayed a much more homogeneous distribution of cells throughout the construct.
Despite a trend for decreasing cell number due to bioreactor culture, use of a bioreactor is vitally important
for improving the homogeneity of tissue engineered constructs and thus overcoming a major setback to
tissue engineering and moving closer to the goal of developing a successful tissue-engineered construct for
use as a bone graft substitute.
Acknowledgements
Irish Research Council for Science, Engineering and Technology
SFI President of Ireland Young Researcher Award
Enterprise Ireland
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